We have chosen to concentrate on a thorough understanding of the oldest family of anti-malarials, the antifolates. Our work in the last few years has shown that a thorough understanding of why and how resistance to these inhibitors is selected can extend the useful therapeutic life of currently available antifolate drugs like pyrimethamine. More important, these studies have also shown that a third generation antifolate, WR99210, holds great promise as an affordable antimalarial that will be effective over a long period. It is effective even against the most pyrimethamine resistant mutants of P. falciparum that have been identified, and mutants resistant to WR99210 have not been easily selected. It is the "low hanging fruit" that drug developers often seek! We propose in this grant to determine the biochemical, genetic and biological mechanisms of action that distinguish WR99210 from pyrimethamine and other antifolate drugs that have been previously developed. * We will analyze the biochemical parameters of mutant enzymes that are resistant to DHFR inhibitors. * We will analyze the fitness of mutant alleles of dhfr in P. falciparum and P. vivax in vitro * We will analyze the changes in pools of mRNA, proteins and small molecules to determine the changes that characterize treatment of P. falciparum with inhibitors of DHFR, with sulfa drugs and with both drugs in synergistic combination. We think that this in depth understanding of antifolate action in the parasites will ensure that WR99210 will be developed as a useful drug for the long haul against P. falciparum and P. vivax.